Evaluation of Somatic Hypermutation Status in Chronic Lymphocytic Leukemia (CLL) in the Era of Next Generation Sequencing.

Sanjeev Kumar Gupta,David S Viswanatha,Keyur P Patel

doi:10.3389/fcell.2020.00357

Abstract

Somatic hypermutation (SHM) status provides an important prognostic indicator for chronic lymphocytic leukemia (CLL), a very common type of mature B-cell leukemia. Owing to the adverse prognosis associated with an unmutated immunoglobulin heavy chain variable (IGHV) status, SHM testing is performed as a standard of care in CLL. Conventionally, SHM testing has been performed using labor intensive and primarily analog Sanger sequencing method following PCR amplification of the clonal immunoglobulin heavy chain gene rearrangements in CLL cells. In comparison, recent availability of next generation sequencing (NGS) allows more versatile detection and direct identification of clonal immunoglobulin gene rearrangements in neoplastic B-cell populations. The ability to identify specific clonal IGHV signature(s) in both baseline (diagnostic) and post-treatment settings enables unique clinical applications of NGS such as determination of SHM status, minimal residual disease (MRD) monitoring, clonal heterogeneity and B cell receptor IG stereotypy. We provide a review of current practices and recommendations for SHM determination using NGS including examples of difficult cases.

Highlights

immunoglobulin heavy chain variable (IGHV) Rearrangements and Somatic Mutations in chronic lymphocytic leukemia (CLL)The IGH region genes encode the antigen binding variable (V) and isotype-specific constant (C) regions of immunoglobulin (IG) heavy chain proteins and their combinations generate tremendous sequence diversity that is important to effectively identify a variety of antigens (Tonegawa, 1983)
The framework region 1 (FR1)/CDR1, FR2/CDR2 and FR3 regions are encoded by the IGHV gene, whereas the CDR3 is formed from the combination of the 3 part of the IGHV gene, the next generation sequencing (NGS)-Based SH Testing in CLL
In contrast to PCR templates produced by leader primers for example, the complete IGHV region cannot be assessed with FR1 primers as ∼60 nucleotide bases upstream of the FR1 primer sites are not sequenced; the germline identity percentage using FR1 anchored primers is a close approximation compared with leader PCR results (Marks et al, 1991; Kuppers et al, 1993)

Summary

IGHV Rearrangements and Somatic Mutations in CLL

The IGH region genes encode the antigen binding variable (V) and isotype-specific constant (C) regions of immunoglobulin (IG) heavy chain proteins and their combinations generate tremendous sequence diversity that is important to effectively identify a variety of antigens (Tonegawa, 1983). A variable number of “non-templated” nucleotides (N) and palindromic (P) nucleotides are added at the V-D and D-J gene junctions, substantially increasing sequence diversity As such the CDR3 sequence represents a highly unique region of the rearranged IGH gene for any given B-cell. IGH gene rearrangements in B-cells undergo additional somatic mutations on exposure to antigen as part of the germinal center reaction in lymphoid tissues. These acquired or somatic hypermutations (SHM) are mediated by the enzyme activation induced cytidine deaminase (AICD) and mainly involve nucleotide base changes in the CDR regions. CLL cases having IGHV3-21 rearrangements ( those belonging to subset #2) are an exception in that these patients have a worse prognosis regardless of SHM status (Tobin et al, 2002; Thorsélius et al, 2006; Baliakas et al, 2015)

Somatic Hypermutation Testing Using Sanger Sequencing

Read Length

Sequencing Platform

Interpretation and Reporting

Findings

AUTHOR CONTRIBUTIONS